The human retinoblastoma gene (RB) is a prototype of a class of growth suppressor genes the inactivation of which contributes to the development of tumors. Existing evidence suggests that the activity of the RB protein is controlled at the level of phosphorylation. Underphosphorylated RB protein present in the GO/G1 phase actively suppressing cell proliferation. Transition from G1 to S and throughout S, the RB protein is inactivated by hyperphosphorylation. The phosphorylation of the RB protein is used as a model to understand how growth suppressor genes are controlled. Our preliminary studies showed that an enzyme preparation that can inactivate the RB protein by phosphorylate is composed of cyclin A and p34 (or antigenically similar molecules) as the main component of the enzyme complex. There appear to be two groups of phosphorylation sites on the RB protein, with one being functionally important (critical phosphorylation site 'C'). The phosphorylation of the critical phosphorylation site 'C' correlated with the inability of the phosphorylated RB protein to bind to SV40 large T antigen. We further showed that the phosphorylation of the critical phosphorylation site 'C' depends on the modification of a binding domain site 'B' on the RB protein. We further showed that site 'B' is a region that conserved among several growth suppressor gene products including RB, p53, and pl07. This suggests a mechanism whereby several different growth suppressor gene products can be coordinately inactivated at specific phases of the cell cycle. Furthermore, the critical site 'C' was not phosphorylated in cells treated with TGF-Beta . These data suggest TGF-Beta may have a direct effect on the modification of the binding site 'B' on the RB protein such that the Rb kinase can not bind. In the present grant proposal, emphasis is placed on understanding how the binding of the RB kinase to the conserved domain site B is controlled by negative and positive growth regulators. We postulate that the positive and negative regulators may all work through the modification of this conserved binding site. Specifically, we propose that positive -regulators convert the binding site 'B' into a form that can be approached by the Rb kinase enzyme complex, whereas the negative regulators either reverse or prevent this process, preventing the Rb kinase enzyme complex from binding various growth suppressor gene products.